Mixed Lineage Kinases (MLKs) are a family of mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) that activate multiple MAP kinase (MAPK) signaling pathways to elicit specific cellular responses. The MLK subfamily consists of MLKs 1, 2, 3, 4 and 4. Of these, MLK3 has been the best characterized in terms of biological function and biochemical activity. MLK3 activates ERK, JNK and p38 MAPK pathways and has important functions in cell proliferation and tumor cell invasion. In contrast, very little is known about the biological functon and biochemical activity of MLK4, for which there are two alternatively spliced isoforms. Our preliminary results suggest that MLK4 does not function as a typical MAPK in promoting activation of MAPK signaling pathways. Instead MLK4 is a negative regulator of MAPK signaling and tumor cell invasion. Furthermore, we have identified a novel interaction between MLK4 and MLK3, and we propose that MLK4 suppresses MAPK signaling and invasion by directly inhibiting MLK3 activation. The experiments described in this proposal are designed to investigate the function of MLK4 in regulating MLK3 activation, MAPK signaling and tumor cell invasion. Accordingly, the specific aims of this proposal are as follows: (1) To analyze the role o MLK4 in regulating MAPK signaling. In vitro kinase assays will be performed to test if MLK4 has activity towards MAP2Ks, and if MLK4 inhibits the activity of other MAP3Ks in addition to MLK3. (2) To investigate the MLK3-MLK4 interaction and the mechanism by which MLK4 inhibits MLK3 activation. In vitro binding assays will be performed with MLK4 and MLK3 deletion mutants to identify the regions of MLK3 and MLK4 that interact. Co-immunoprecipitations will be performed in cells overexpressing MLK4 to determine if MLK4 inhibits the MLK3-TRAF, MLK3-Cdc42 or MLK3-B-Raf interactions. (3) To investigate the function of MLK4 in tumor cell proliferation and invasion. Cell proliferation assays and invasion assays (using a modified Boyden chamber) will be performed to test if MLK4 expression or knockdown affects cell proliferation or invasion. The results from the proposed studies will broaden our understanding of the different roles of MLKs in cell signaling and tumor cell invasion, which could provide insight into how altered MLK activity may contribute to establishment of the malignant phenotype. PUBLIC HEALTH RELEVANCE: MAPK signaling pathways have a central role in the regulation of numerous cellular responses such as proliferation, differentiation, migration and invasion. Since persistent activation of MAPK signaling is a major contributor to the establishment of a malignant phenotype, a thorough understanding of how MAPK pathways are tightly controlled by both positive and negative regulatory factors is essential to the development of novel cancer therapies.